Specifically, many diseases are now understood at the molecular and genetic level. Analysis of such molecules is important for disease diagnosis and prognosis. Current methods for direct extraction of cellular tissue material from a tissue sample are limited because the extraction reflects only the average content of disease associated markers. In reality, tissues are very heterogeneous and the most diagnostic portions of the tissue may be confined to a few hundred cells or less in a lesion. Thus molecular analysis of human pathology sections will require the targeting and removal of pure populations of homogeneous cells from within a specimen where these cells may comprise only a few percent of the total local tissue or even much less (e.g., 1 cell in a 1000 or even less). The modification of the LCM invention covered in this application is particularly important in the collection of sparse or rare cells within a tissue and cases where such cells must be collected from a variety of specimens in order to provide sufficient material for an accurate statistical representation of the pathology. Examples include isolation cells that were infected by an AIDS virus, macrophages containing an infectious tuberculosis bacteria, cells within a kidney glomerulus in patients exhibiting proteinuria, brain cells and plaques in brain exhibiting Alzheimer's Syndrome, as well as cancerous tissues. In each case the precise molecular description of the variant of the disease which might be related to different natural etiology and responses of that particular patient to different therapies can only be accomplished without gross contamination of molecules from surrounding cells. Though we specifically discuss how laser microdissection is critical in the molecular analysis of cancer, the same principles apply to a molecular description (e.g., DNA mutations, alterations of gene expression, and post-transcriptional modification of proteins) of a variety of diseases and their response to drug treatment and of normal human developing and aging.
Normal tissue samples contain a variety of cell types surrounding and adjacent to the pre-invasive and invasive tumor cells. A region of the tumor tissue subject to biopsy and diagnosis as small as 1.0 mm can contain normal epithelium, pre-invasive stages of carcinoma, in-situ carcinoma, invasive carcinoma, and inflammatory areas. Consequently, routine scraping and cutting methods will gather all of these types of cells, and hence, loss of an allele will be masked by presence of a normal copy of the allele in the contaminating non-malignant cells. Existing methods for cutting away or masking a portion of tissue do not have the needed resolution. Hence the analysis of genetic results by those previous methods are always plagued by contaminating alleles from normal cells, undesired cells or vascular cells.
The molecular study of human tumors is currently limited by the techniques and model systems available for their characterization. Studies to quantitatively or qualitatively assess proteins or nucleic acid expression in human tumor cells are compromised by the diverse cell populations present in bulk tumor specimens. Histologic fields of invasive tumor typically show a number of cell types including tumor cells, stromal cells, endothelial cells, normal epithelial cells and inflammatory cells. Since the tumor cells are often a relatively small percentage of the total cell population it is difficult to interpret the significance of net protein or nucleic acid alterations in these specimens.
Studies of human tumor cells in culture do not account for the complex interactions of the tumor cells with host cells and extracellular matrix, and how they may regulate tumor cell protease productivity or activation. Immunohistochemical staining allows one to examine enzyme distribution in regions of tumor invasion, however, results vary with tissue fixation and antibody-antigen affinity, and provide only a semi-quantitative assessment of protein levels. Furthermore, quantitative interpretation of staining results is complicated by the variability of staining patterns within tissue sections, subjective evaluation of staining intensity, and the difficulty in interpreting the significance of stromal staining. In addition, many antibodies utilized in the study of proteases do not differentiate pro-enzyme from active enzyme species. Assays of enzyme or mRNA levels from homogenates of human tumors does not account for either the mixed population of cells within the specimens, or the concomitant pathophysiologic processes which may occur in the tissue.
Prior methods of study have not allowed investigators to specifically examine genetic alterations in pre-invasive lesions. Even the most sophisticated genetic testing techniques to date have been of limited value because the input DNA, RNA or proteins to be analyzed are not derived from pure cell populations exhibiting the disease morphology. Several methods have been reported for tissue microdissection to address this problem. These include gross dissection of frozen tissue blocks to enrich for specific cell populations, irradiation of manually ink stained sections to destroy unwanted genetic material, touch preparations of frozen tissue specimens and microdissection with manual tools. These methods, however, are not sufficiently precise and efficient for routine research or high throughput clinical molecular diagnostic applications. Manual microdissection, for example, has good precision but is time consuming, labor intensive, requires a high degree of manual dexterity, and is not generally suitable for the ordinary technologist.
In Lance A. Liotta et al. U.S. Provisional Patent Application Serial 60/036,927 filed Feb. 7, 1997, entitled Isolation of Cellular Material Under Microscope Visualization, there is described a technique that we have come to call Laser Capture Microdissection (LCM). Simply stated, a method and apparatus was disclosed in which a tissue sample was provided, typically on a slide under observation in a microscope. The tissue was contacted with a selectively activated surface which could be activated to provide selective regions thereof with adhesive properties. The tissue sample is visualized through a microscope and at least one portion of the tissue sample which is to be extracted is identified. Thereafter, the selectively activated surface is activated, typically by a laser routed through a fiber optic being directed onto the selectively activated surface in the footprint of the desired tissue. This is done while a region of selectively activated surface is in contact with the portion of the tissue sample selected. The activated region of the selectively activated surface adheres to that portion of the tissue sample. Thereafter, the activated surface is separated from the tissue sample while maintaining adhesion between the activated region of selectively activated surface and the portion of the tissue sample. The portion of the tissue sample is extracted from the remaining portion of said tissue sample.
While the basic technique disclosed in the above-entitled patent deals with applying a large free piece of EVA film to a tissue sample on a slide, the reduction of this technique to a practical method and apparatus which can be utilized by relatively untrained personnel with conventional microscopes is not set forth. Accordingly, in the following specification, one practical embodiment of this technique is set forth.